ABSTRACT Regulatory T cells accumulate and correlate with poor prognosis in many cancers, and ablation of Treg together with other therapies is being explored in the clinic. The success of this approach requires, however, elucidation of the mechanisms of regulation and action of Treg in order to provide new, more selective targets for rational immunotherapy. This proposal derives from our discovery of a novel Treg-intrinsic signaling pathway required for contact-dependent suppression of tumor immunity. In this pathway, Treg-expressed CTLA4 physically associates with protein kinase C-eta (PKC?) and, upon CTLA4 engagement, recruits a PAK- PIX-GIT complex required for focal adhesion disassembly. PKC? deletion or expression of a CTLA4 non- interacting PKC? mutant impaired Treg ability to inhibit tumor-specific immunity, but not autoimmune colitis. We hypothesize that this novel CTLA4 signaling axis is obligatory for contact-dependent suppression of tumor immunity by Foxp3+ Treg. Our overarching goal is to further explore at the mechanistic and animal levels this signaling pathway and its functional implications for tumor immunity. In Aim 1, we will mechanistically analyze the molecular pathways utilized by CTLA4 and PKC? to recruit and activate the GIT2-PAK2-?PIX complex and mediate contact-dependent suppression in mouse and human Treg, and determine how disruption of this signaling pathway affects Treg suppression. We will also use 2-photon microscopy to study intratumoral Treg- DC dynamics. Given the critical role of CD8+ T cells as tumor-killing CTL, we will also determine how CD8- specific PKC? deletion affects their activation and functions. In Aim 2, we will use mice with constitutive, Cre- mediated deletion of PKC? (and GIT2) in Foxp3+ Treg to analyze in vivo how disrupting CTLA4-PKC? signaling affects tumor immunity and the tumor microenvironment. We will use preclinical tumor models, including a melanoma carrying the BrafV600E mutation and a genetically engineered mouse hepatocellular carcinoma model. We will focus on tumor-infiltrating Treg, CD8+ cells and DC, and explore the transendocytosis pathway of Treg-mediated depletion of costimulatory CD80/CD86 ligands from antigen- presenting cells, which is impaired in Prkch?/? Treg. If Aim 1 studies reveal an important role for PKC? in CD8+ T cells, we will additionally analyze tumor growth and the tumor microenvironment in mice with CD8-specific Prkch deletion. In Aim 3, we will use mice with a tamoxifen (Tam)-induced, time-controlled Treg-specific PKC? deletion, either alone or in combination with adjunct therapies, to study the therapeutic effects of these manipulations, using the tumor models in Aim 2. The studies will benefit from availability of all tools and models and from expert collaborators. We expect this project to provide mechanistic understanding of a novel pathway that Treg utilize to suppress tumor-specific immunity and promote tumor growth. This understanding could lead, in turn, to novel cancer immunotherapies based on selective inactivation of the CTLA4-PKC? signaling pathway in Treg, resulting in inhibition of cancer-promoting Treg with minimal autoimmune-related side effects.